Physical and Biological Study of Psilocybe cubensis Mushrooms and Their Psilocybin and Psilocin Compounds

The Psilocybe cubensis mushrooms used in this study were imported into Brazil (Rosehill Apothecary Ltd., Rose Hill, Jamaica) through an import permit granted by ANVISA (AI 067/2025), specifically for this project, in accordance with the Special Authorization for the study of psilocybin and psilocin (AE No. 30/2024), which includes the control, storage, and handling of these substances. The mushrooms were dried by the supplying company and delivered in hermetically sealed packaging. Psilocybin (1 mg/mL, Lipomed Document QC-CA-411L1) and psilocin (1 mg/mL, Lipomed Document QC-CA-410L1) analytical standards were obtained from LAS do Brasil (Aparecida de Goiânia, Goiás, Brazil). HPLC-grade acetonitrile (ACN) was acquired from Êxodo Científica (Sumaré, São Paulo, Brazil) and SK Chemicals (Seongnam, South Korea), whereas analytical grade formic acid (P.A.) was sourced from Neon (Suzano, São Paulo, Brazil). Ultrapure water (18.2 MΩ·cm, TOC < 10 ppb) was produced via a Milli-Q system (MERCK, model eq 7000, Darmstadt, Germany).

Scanning electron microscopy (SEM) was performed to analyze the morphology of the Psilocybe mushrooms. The mushroom was sectioned into three parts: the upper region of the cap (fraction 1), the gills located on the underside of the cap (fraction 2), and the stipe or stem (fraction 3), which were analyzed at magnifications of 250× and 1000× (Figure). From the micrographs, it is possible to observe that fraction 1 has a rough surface profile, likely due to mushroom dehydration. At a magnification of 1000×, microfibers and light-colored particles can be observed, which may be associated with the mineral components present in the mushroom. In the gill region (fraction 2), at 250× magnification, a smooth surface with scattered dots is observed. When magnified to 1000×, these structures are identified as mushroom spores, some of which appear ruptured, whereas others remain intact, displaying a morphology similar to that of red blood cells. Analysis of the stipe revealed a fibrous appearance, with fibers oriented in the direction of mushroom growthcharacteristic of the stems of plants and fungal materials.

The particle morphology of the mushroom powder was analyzed at 250× magnification, along with elemental composition analysis via energy-dispersive X-ray spectroscopy (EDS), in order to estimate the approximate elemental content, with a particular emphasis on phosphorus, an element present in the chemical structure of psilocybin, as shown in Figure. The micrographs revealed particles of varying sizes, with the powder sample exhibiting predominantly rough and fibrous surfaces, corresponding to the cap and stipe regions, respectively. The EDS spectrum displays ionization energy and counts, where higher counts indicate greater presence of a given element. On the basis of the SEM–EDS micrograph, the predominant chemical elements were found to be spatially distributed throughout the analyzed section, with carbon, potassium, oxygen, and phosphorus highlighted in red, purple, green, and blue, respectivelyelements commonly associated with fungal matrices. Additionally, trace amounts of chlorine, magnesium, iron, aluminum, and silicon were also detected. Although the elemental composition of hallucinogenic mushrooms has not been widely reported in the literature, these mushrooms can be analyzed similarly to edible mushrooms, allowing for the mapping and identification of major chemical constituents.

Studies on edible mushrooms have reported the presence of elements such as calcium, iron, magnesium, phosphorus, potassium, sodium, zinc, copper, manganese, and selenium.However, in the present study, energy-dispersive X-ray spectroscopy (EDS) analysis did not detect calcium, sodium, zinc, copper, manganese, or selenium. Additionally, previously listed elements, including chlorine, aluminum, and silicon, were identified. The presence of chlorine may be attributed to phenolic compounds, such as pentachlorophenol.Muñoz, Corona, Wrobel, Soto and Wrobelinvestigated the subcellular distributions of aluminum, bismuth, cadmium, chromium, copper, iron, manganese, nickel, and lead in cultivated mushrooms, and reported the presence of aluminum localized in the cell walls of certain species. Silicon, on the other hand, has not been widely documented in the literature, although it is a common component of sand and may appear as a contaminant introduced during cultivation, harvesting, storage, or processing. Although EDS elemental analysis provides useful insights into the spatial distribution of key elements, complementary analytical techniques are necessary for more accurate characterization. ,

Macronutrients such as carbon (C), hydrogen (H), sodium (Na), potassium (K), chlorine (Cl), and calcium (Ca), among others, are typically present at concentrations in the percentage range or in milligrams per gram (mg/g). Micronutrients, or trace elements, such as iron (Fe), zinc (Zn), selenium (Se), and cobalt (Co), among others, are found at significantly lower concentrations, typically in the microgram per gram (μg/g) to nanogram per gram (ng/g) range.Figure shows the X-ray fluorescence (XRF) spectrum obtained from the atomic ionization of the elements detected in the powdered sample of Psilocybe genus mushrooms. Table presents the elemental analysis and the corresponding percentage compositions of the identified elements.

According to the obtained data, the Psilocybe mushroom contains a variety of identified chemical elements, including magnesium, phosphorus, sulfur, chlorine, potassium, manganese, iron, copper, zinc, bromine, rubidium, tin, tellurium, and osmium. Mineral composition analysis revealed the predominant presence of potassium oxide (61.7%) and phosphates (P₂O₅ at 24.33%), also confirmed by EDS analysis. These were followed by sulfur trioxide (7.7%), chlorine (3.9%), magnesium oxide (1.7%), and other elements present at concentrations below 1%. The identified elements do not exhibit inherent toxicity; however, manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) may be potentially toxic if they are consumed in excessive amounts. The remaining elements (Mg, P, S, Cl, K, Br, Rb, Sn, Te, and Os) are essential to human physiology when present at appropriate concentrations and are generally not considered toxic at normal dietary levels, indicating that the sample is nontoxic. The detection of P₂O₅ suggests the presence of psilocybin in the mushroom, highlighting its potential for the extraction of psilocybin with yields suitable for the development of active pharmaceutical ingredients (APIs) based on this compound.

An investigation of wild medicinal mushrooms of the species Ganoderma lucidum by Popa et al. revealed the presence of the chemical elements Si, P, Fe, Ag, Ca, K, Mn, Sr, S, and Ni in most of the collected samples. However, a wide range of additional elements was also detected, including Ag, Al, As, At, Br, Ca, Cl, Co, Cu, Eu, Fe, Fr, Ga, Gd, Ge, Ir, K, Lu, Mn, Mo, Mg, Ni, Os, P, Pb, Po, Pt, Rb, Re, S, Se, Si, Sr, Ti, Tm, U, V, W, Y, and Zn, highlighting the compositional variability among mushrooms. The most significant mineral content observed in this species included iron and/or potassium. In a similar study on wild mushrooms, Turhan et al. quantified the levels of K, Fe, Cu, Mn, Zn, Pb, Cd, Ni, Sn, Br, Sr, Ti, Rb, As, Th, and U across 11 species of wild mushrooms. Among all the analyzed samples, not all contained elements such as zinc, cadmium, nickel, selenium, bromine, rubidium, arsenic, thorium, and uranium. Potassium was consistently the most abundant element, which is in agreement with the findings of the present study. However, iron was reported as the second most abundant element in Turhan's study, which contrasts with the results obtained for Psilocybe, where the iron content was notably lower in comparison.

The powdered Psilocybe mushroom and the obtained active pharmaceutical ingredient (API) were analyzed via simultaneous thermal analysis (STA), and Fourier transform infrared (FTIR) spectra of the evolved gases, which were collected during sample heating. On the basis of thermogravimetric analysis (Figure), which is supported by its derivative curve, five distinct mass loss events can be identified. The first mass loss, observed in the TGA curve (black line), occurred between 30–117 °C, with a maximum at 73 °C, as indicated by the DTG peak (red line). The second event occurred between 117–162 °C, with a peak at approximately 140 °C, which corresponds to the elimination of adsorbed water bound to the matrix and physically retained moisture, despite the samples being dried. The third mass loss takes place between 162–217 °C, with a maximum at 200 °C, that is associated with the volatilization of low-molecular-weight compounds. The fourth event, between 220–375 °C, with a peak at 307 °C and a shoulder at 338 °C, corresponds to the pyrolysis of organic components, which is related primarily to polysaccharides and microbial cell wall constituents, especially cellulose and hemicellulose, as well as the release of more thermally stable volatiles. Finally, the fifth mass loss begins at approximately 375 °C and extends to approximately 500 °C, with a noticeable shoulder, and is attributed to the combustion of macromolecular cellulose derivatives and the presence of thermally resistant compounds such as lignin.

As observed by Sharma, mass loss during the thermal degradation of mushroom constituents can be attributed to the successive combustion of components such as cellulose, xylan, chitin, microbial biomass, and phenolic compounds. The author anticipated thermal responses between 200–420 and 420–580 °C, corresponding to primary and secondary peaks, respectively, as seen in the DTG curve (Figure). The results revealed water loss at approximately 90–110 °C, a major weight loss between 200–420 °C associated with hemicellulose and microbial cell walls, a minor peak near 420 °C attributed to structural hemicellulose, and a secondary activity peak between 420–580 °C linked to the shoulders or decomposition of more stable compounds, such as lignin. These findings are consistent with those observed for the Psilocybe mushroom in the present study. However, while Sharma identified only one water-related mass loss event, two distinct losses were observed here, possibly influenced by different heating rates during analysis. Ming et al. evaluated the thermal properties of Lentinus edodes mushrooms and identified three mass loss stages via thermogravimetric analysis: the first stage occurred between 35–200 °C, the second stage occurred between 200–350 °C, and the third stage occurred between 350–590 °C. The first was related to the loss of adsorbed water and the decomposition of thermolabile compounds, the second was related to the oxidative degradation of organic matter and volatile components, and the third was related to the combustion of cellulose macromolecules. The mass loss events reported by these authors are in agreement with those identified in the present study.

On the basis of DSC analysis Figure, five thermal events were identified: the first two peaks are endothermic, followed by three exothermic events. The first endothermic event begins at approximately 30 °C and extends to 120 °C, with a peak temperature at 76 °C and an enthalpy change (ΔH) of 93.04 J/g, corresponding to the release of adsorbed water. The second endothermic transition occurs between 172–225 °C, peaks at 203 °C, with an enthalpy of 11.09 J/g, and is associated with the volatilization of low-molecular-weight compounds. The subsequent exothermic events occurred in the ranges of 236–266, 290–372, and 437–646 °C, with peak temperatures at 252, 335, and 551 °C, and enthalpy changes of ΔH = −3.44, ΔH = −35.14, and ΔH = −267.10 J/g, respectively. These exothermic peaks correspond to the thermal decomposition and degradation of organic constituents. The events between 250–400 °C are attributed mainly to the decomposition of cellulose and hemicellulose, whereas the degradation of lignin occurs at temperatures above 400 °C, characterized by continuous mass loss.

The particle processing conditions of Tremella fuciformis mushroom powder were investigated by Tsai et al. These researchers reported that smaller particle sizes provided greater thermal stability, as evidenced by DSC and TGA analyses. The endothermic events identified by the authors involved varying onset temperatures, reflecting the influence of particle size on the hydration behavior and thermal stability of the samples. However, only one thermal event of each type (endothermic or exothermic) was reported. In comparison, the peak temperature of the endothermic event observed in the present study was lower than the range, with a smaller enthalpy requirement, suggesting a reduced water content in the sample. Moreover, the volatilization event was not detected by those authors, possibly because of the co-occurrence of moisture loss and volatilization at lower temperatures, resulting in a single thermal event. With respect to the exothermic event, the peak temperature of 252 °C found in this study was within the range observed by Tsai et al., but the enthalpy was significantly lower. Additionally, three distinct exothermic events were identified here, likely due to the compositional variability of the organic constituents in the material.

On the basis of the STA analysis, infrared spectra were collected at the characteristic temperatures corresponding to the mass loss peaks observed in the TGA/DTG curves and the thermal events identified via DSC in order to elucidate the structures of the volatilized substances, as shown in Figure. The analysis of gases released during thermal events, conducted via thermogravimetric analysis coupled with FTIR spectroscopy, enabled the identification of volatile compounds emitted throughout the thermal degradation process of both whole mushrooms and their extracts. This integrated approach, which combines TGA/DTG/DSC data with FTIR spectral information, provides a comprehensive understanding of the thermal and chemical transformations occurring within these materials.

In the mushroom powder, the initial mass loss observed between 30–117 °C, with a maximum at 75 °C, was associated with the release of adsorbed and chemically bound water within the material matrix. This event was corroborated by the presence of an infrared absorption band at approximately 1640 cm^–1, corresponding to the bending vibration of water molecules. Alongside the second mass loss, which occurs between 117–162 °C and peaks near 140 °C, the continued presence of water, both physically adsorbed and chemically bound, was confirmedalthough with a reduced intensity compared with that of the previous stage. In this interval, new absorption bands emerged within the 1750–1850 cm^–1 and 2250–2400 cm^–1 regions, indicating the onset of thermal decomposition of volatile organic compounds or residual carbonyl-containing groups, such as aldehydes and ketones. The FTIR spectrum associated with the third mass loss event, observed at 202 °C in the analysis of the whole mushroom, revealed more defined bands in the 2800–3000 cm^–1 range, corresponding to the stretching vibrations of C–H bonds in methyl and methylene groups, suggesting the release of volatile compounds such as alcohols and low-molecular-weight hydrocarbons. Concurrently, the bands in the 1700–1750 cm^–1 region became more pronounced, suggesting further elimination of aldehydes and ketones. As the temperature increased (approximately 260 °C), characteristic polysaccharide bands began to dominate the spectra, particularly within the 1000–1200 cm^–1 range, corresponding to C–O stretching vibrations linked to sugar decomposition. Additional bands in the 1500–1600 cm^–1 region indicated the formation of aromatic compounds, likely arising from the thermal breakdown of cell wall components such as cellulose and hemicellulose. In higher temperature ranges (310.60 and 353.88 °C), the spectra exhibited a sharp increase in absorption bands within the 2300–2400 cm^–1 region, attributed to CO₂ evolutiona major combustion product of thermally stable organic matter such as lignin. Persistent bands in the 1500–1600 cm^–1 region remained evident and were associated with the degradation of complex aromatic structures. This thermal behavior supports the progressive combustion of organic residues, highlighting the relatively high thermal stability of lignin.,

Like the thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) results obtained for the mushroom powder, the analysis of the mushroom extract (Figure) enabled the identification of several thermal events related to its chemical composition and thermal behavior. The TGA curve revealed three major stages of mass loss for the extract. The first mass loss occurred within the temperature range of 90–230 °C and was characterized by a prominent DTG peak at approximately 110 °C, accompanied by a set of lower-intensity peaks throughout this interval. This event is attributed to the release of residual water, including both physically adsorbed and chemically bound water, as well as the volatilization of residual compoundspossibly related to the presence of secondary metabolites or solvent residues from the extraction processwhich were less evident in the analysis of the raw mushroom material. While this thermal behavior resembled that observed in the raw sample, the transition in the extract appeared more concentrated, indicating that the extraction process reduced the heterogeneity of the material matrix. Additionally, previous studies reported the volatilization of phenolic compounds and terpenes within this temperature range, further supporting the interpretation of this thermal event.−

The second mass loss occurred between 240–400 °C, with a prominent DTG peak at 300 °C and a secondary peak at 312 °C. This thermal event is associated with the degradation of organic compounds, particularly polysaccharides extracted from mushroom cell walls. Previous studies have reported that constituents such as cellulose and hemicellulose undergo significant thermal decomposition within this temperature range.Furthermore, the DSC curve (in blue) displays an exothermic peak in this interval, supporting the occurrence of structural component degradation. The third stage of mass loss takes place between 400–600 °C, with a shoulder at approximately 450–500 °C, indicative of the combustion of thermally stable macromolecules such as lignin and other recalcitrant phenolic compounds. The presence of these compounds is also evident in the analysis of the raw mushroom sample but appears to be more concentrated in the extract, suggesting that the extraction process enriched the content of these thermostable constituents. Finally, the residual mass observed above 600 °C corresponds to the mineral fraction or ash content, representing the inorganic compounds present in the extract.

DSC analysis complements this interpretation by revealing both endothermic and exothermic transitions. The endothermic peak near 100 °C is attributed to water evaporation, whereas the exothermic events observed at approximately 350 and 450 °C are associated with the degradation and combustion of organic compounds. The absence of more pronounced endothermic peaks above150 °C suggests that the compounds present in the extract exhibit high thermal stability or were already released during earlier stages. To gain a better understanding of the substances volatilized during the thermal process, the FTIR spectra of the compounds released at the peak temperatures of the thermal events were analyzed, as shown in Figure.

In the initial temperature range of 120 °C, a band between 1720–1840 cm^–1 is observed, which is typical for C=O stretching vibrations. This suggests the release of volatile aldehydes, ketones, or carboxylic acids. In the region between 1130–1231 cm^–1, corresponding to C–O bonds frequently observed in alcohols, ethers, or ester-containing compounds, the spectra indicate the initial decomposition of polysaccharides or other extract components containing oxygen bonded to carbon chains. This behavior differs slightly from that of the whole mushroom, where the dehydration of adsorbed water dominates the early thermal events. As the temperature increased to 143 °C, the bands at approximately 3000, 1700–1750, and 1000–1200 cm^–1 remained highly intense, confirming the emission of organic compounds derived from the initial decomposition of molecules present in the extract. At 165 and 186 °C, there was an intensification of the bands in the 2800–3000 cm^–1 region, confirming the release of aliphatic hydrocarbons. More defined bands in the 1700–1750 cm^–1 range indicate the liberation of carbonyl compounds, whereas the absorptions between 1000–1200 cm^–1 reflect C–O vibrations characteristic of alcohols and esters. Upon reaching 293 °C, the band in the 2300–2400 cm^–1 region becomes significantly intensified, characterizing the predominant release of CO₂a typical product of carbohydrate thermal decomposition. In this interval, the absorption in the 1500–1600 cm^–1 range indicates the presence of aromatic compounds, suggesting the onset of lignin degradation as well as the breakdown of other thermostable components. The residual bands in the 1000–1200 cm^–1 region still reflect the decomposition of the remaining polysaccharides. Finally, in the temperature range of 312 °C, the band in the 2300–2400 cm^–1 region dominated, reflecting the massive release of CO₂ during the combustion of thermostable macromolecules.,

Compared with the raw mushroom, the extract exhibited a more clearly defined profile of thermal events, reflecting the concentration of specific compounds present during the extraction process. In whole mushrooms, the release of adsorbed water occurs at slightly lower temperatures, indicating lower moisture retention than in the extract. Furthermore, the presence of polysaccharides and lignin is more pronounced in the whole mushroom, as demonstrated by the higher intensity of bands related to C–O vibrations and CO₂ emissions at intermediate temperatures, revealing the characteristics of a more heterogeneous matrix with a broader range of mass loss temperatures. In contrast, in the extract, the increased intensity of bands in the 1700–1750 cm^–1 region reflects the concentration of volatile and bioactive compoundspossibly enriched during the extraction processwhich characterize it as a more homogeneous material, thereby facilitating the identification of volatile compounds, polysaccharides, and lignin.

The qualitative phytochemical analyses (Table) of the mushroom in powder form revealed the following: saponins were detected by the formation of foam; polysaccharides were evidenced by a blue color shift; tannins and phenolic compounds were indicated by the appearance of a blue coloration; flavonoids were identified exclusively through the oxalo-boric reaction, with fluorescence observed under ultraviolet light; and alkaloids yielded positive results in all tests, as demonstrated by a color change in the Bouchardat and Dragendorff reactions, along with the formation of a white precipitate in the Mayer test. Further, steroids and terpenoids were identified on the basis of the observed color changes. No traces of other metabolites, such as quinones or coumarins, were detected. In the fungal extract (API), only the steroids, terpenoids, and saponins were not extracted; that is, the Psilocybe mushroom extract contains tannins, phenolic compounds, flavonoids, alkaloids, and polysaccharideswith the indication of polysaccharides showing only a slight color change, suggesting a low concentration of these compounds.

The presence of alkaloids, tannins, phenolic compounds, polysaccharides, and flavonoids in the extract confirms the occurrence of bioactive principles that may exhibit medicinal properties. Psilocybin and psilocin are alkaloids; therefore, the extraction method used in this research is assumed to be efficient in isolating the target substances. These results agree with the findings of Dhanasekaran et al., who conducted a taxonomic identification of bioactive compounds in Psilocybe mushrooms. They analyzed the crude extract and observed the presence of saponins, tannins, flavonoids, and alkaloids, differing only with respect to the detection of saponins. Moreover, the authors reported that steroids and terpenoids were not detected, which corroborates the results obtained for the Psilocybe mushroom extract. Margret, Mareeswari, Kumar and Jerley investigated the presence of phytochemicals in extracts of Agaricus bisporus and Pleurotus ostreatus, identifying saponins, tannins, phenolic compounds, terpenes, steroids, flavonoids, glycosides, alkaloids, and amino acids. The only divergence observed was in the identification of steroids, while the presence of the other compounds was consistent with the substances identified in the present study.

The FTIR analysis of the mushroom, as shown in Figure, reveals important information about its molecular composition, particularly regarding the presence of proteins and polysaccharides. The spectrum shows a prominent absorption band between 3400 and 3000 cm^–1, which corresponds to the vibrational contributions of −NH bonds and, primarily, to the symmetric and asymmetric stretching vibrations of −OH groups. These differences are attributed to water molecules and carbohydrate structures. In addition to stretching, water also exhibits an infrared-active bending vibration related to the opening and closing of the H–O–H bond angle, which appears at approximately 1640 cm^–1. This band may overlap with other molecular vibrations, such as the C=O stretching of amide groups. The region between 2900 and 2850 cm^–1 is associated with the stretching vibrations of methyl and methylene groups (CH₂/CH₃), likely originating from polysaccharides and lipids. This is further supported by the absorption near 1150 cm^–1, attributed to glycosidic linkages, as well as the region between 1080–1000 cm^–1.

On the basis of the spectral analysis, a comparative assessment can be made with data reported in the literature to better understand the spectroscopic behavior of the mushroom, as shown in Table, and to establish a comparison with the obtained extract. A comparison of the current data with literature data revealed that the absorption band at 1635 cm^–1 is associated with the amide I vibration, corresponding to the C=O stretching of proteins, which is indicative of the presence of peptide bonds. The band at 1543 cm^–1 corresponds to the amide II vibration, which is also related to protein components. Typically, this frequency appears at a lower wavenumber than the C=O stretching of carboxylic acids or esters, suggesting a low contribution of lipid content in this fungus.

The presence of bands at approximately 1400 cm^–1 is attributed to out-of-phase CH bending and COH bending vibrations, whereas the band at 1368 cm^–1 is associated with in-phase C–H bending. In the region of 1232 cm^–1, the band can be linked to C–C stretching of the pyranose ring and to stretching vibrations of carboxylic acids or ester −CO groups, suggesting the presence of cyclic structures and potential steric interactions. At lower frequencies, approximately 1153 cm^–1, the C–O–C stretching vibration of the glycosidic linkage appears, which is characteristic of polysaccharide structures. A broad and intense band at 1077 cm^–1 is assigned to the −CO stretching of alcohol groups, which are predominantly found in polysaccharides. This band may also encompass contributions from C–C stretching and possibly −POC group vibrations in the presence of trace amounts of psilocybin or lipids. Additionally, a weak band observed at 890 cm^–1 may be related to β-linked polysaccharides, particularly due to C–H deformation or β-glycosidic linkages.

These results indicate that P. cubensis has a complex matrix rich in proteins and polysaccharides, with minimal evidence of lipids or fatty acids, reflecting its biochemical composition and both structural and bioactive functionalities. Compared with the obtained ATR-FTIR spectrum, a similar pattern can be observed, with variations in absorption bands within the 1750–1200 cm^–1 range, corresponding to the amide I and amide II bands of proteins, as well as carboxylic acid and ester vibrations. These findings suggest a limited or absent extraction of these compound classes. Furthermore, the definition of the P–O–C absorption band suggests a higher concentration of compounds containing phosphate ester linkages.

The concentrations of arsenic, cadmium, mercury, and lead were quantified, with reference values provided according to the lowest permissible limits established by the International Council for Harmonization (ICH), and the Brazilian Pharmacopoeia (BP), as shown in Table. On the basis of the obtained results, all four analyzed elements are below the limits specified by both regulatory standards used as a reference for this study, demonstrating the material's purity in terms of heavy metal content for both the raw material (mushroom powder) and the obtained extract (API). Furthermore, the data revealed a reduction in metal content after the extraction process, with cadmium levels reduced by 100% and lead levels reduced by approximately 50%. This suggests that the extraction process does not fully transfer metallic elements to the final product, which is favorable for the intended biomedical applications of the material.

A bibliometric analysis of European publications between 2001 and 2016 conducted by Świsłowski et al. regarding the concentrations of various elements in mushrooms indicated that cadmium was the most frequently discussed element. The highest cadmium concentration reported in Paxillus involutus was 3964 mg/kg. The highest concentration of lead was observed in Amanita citrina, at 895 mg/kg, followed by Macrolepiota procera, at 171 mg/kg. Although these concentrations exceed those reported in the present study, the mushrooms analyzed by the authors were collected from regions with probable environmental, which may account for the variation in phytochemical content. Moura performed heavy metal determination in 24 samples across three mushroom genera (Pleurotus, Lentinus, and Agaricus) via triplicate measurements and calculated average concentrations of multiple elements. Among the four elements previously mentioned, only arsenic was quantified, with concentrations ranging from 0.020 to 0.206 μg/g in Lentinus, 0.094 to 0.402 μg/g in Agaricus, and 0.027 to 0.080 μg/g in Pleurotus. Compared with those in the Psilocybe mushroom powder analyzed in this study, the arsenic levels observed were lower than those. Falandysz et al. carried out an elemental analysis of 38 elements in wild mushrooms from Poland. The authors reported relatively low concentrations of lead (0.54–1.3 μg/g), cadmium (0.56–12 μg/g), and mercury (0.19–5.5 μg/g), with arsenic not being quantified. In comparison, the cadmium content in Psilocybe mushrooms analyzed in the present study was below the minimum level reported by Falandysz and colleagues, lead concentrations were within the same range, and mercury was not detected in our sample.

The microbiological evaluation of the mushroom powder and the obtained IFA extract was based on the enumeration of aerobic microorganisms, yeasts, and molds, following the pour plate method. The results are presented in Table. Microbiological analysis revealed that microbial loads tend to be lower in the material after the extraction process, with a significant reduction in CFUs. The aerobic microorganism count decreased from 5.23 log CFU/mL to values below 1 log CFU/mL, whereas the fungal count decreased from 5.38 log CFU/mL to less than 1 log CFU/mL, indicating that the extracted material contained a markedly lower total microbial burden. Considering that mushroom powder is a raw natural product, the CFU levels observed exceeded the limits established by the Brazilian Pharmacopoeia. However, this material can be classified as untreated or non-preprocessed. In contrast, the API showed microbial counts well below all the pharmacopoeial limits, regardless of the classification criteria, highlighting its potential for biomedical applications under appropriate microbial quality control standards.

In a study conducted by Venturini et al.,402 samples from 22 species of fresh and wild mushrooms were evaluated. The microbial counts ranged from 4.4 to 9.4 log CFU/g, with no significant differences observed among the species. The average values of the yeast population were between 3.2 and 3.7 log CFU/g. Compared with the samples analyzed in the present study, the total microbial count fell within the range reported by the authors, although notably lower than the median values observed. In contrast, the counts for yeasts and molds were relatively high. Kim et al.assessed the microbial load in commercial shiitake mushrooms, identifying aerobic microorganism levels ranging from 3.3 to 7.5 log CFU/g and yeast and mold counts between 2.2 and 6.0 log CFU/g. When compared, the values obtained for the materials analyzed in the present study fall within the ranges reported by these authors.

The cell growth viability of the API was assessed through an in vitro fibroblast (L929) cell assay via the MTT test, which serves as a reliable marker for evaluating the cytotoxicity of bioactive compounds. The cells were cultured and exposed to the API at concentrations of 5, 25, 50, 75, 100, and 1200 μg/mL. The assay was conducted in 12-well plates, and the average standard deviation was calculated, as shown in Figure. Polyphenolic compounds, along with polysaccharides, proteins, and their complexes, may exert cytotoxic effects in mushroom extracts, depending on their concentration, molecular interactions, and extraction conditions., Among these groups, the obtained API demonstrated the presence of unquantified polyphenols and proteins, which may contribute to reduced cell viability. Therefore, it is necessary to evaluate the potential cytotoxicity these constituents may exert on the material.

On the basis of the cell viability results, lower concentrations of the API (<50 μg/mL) resulted in viability levels below 70%, whereas concentrations above this threshold maintained cell viability above 74%. The highest concentration tested (1.2 mg/mL or 1200 μg/mL) resulted in a cell viability profile comparable to that of the other concentrations, indicating the absence of cytotoxic effects even at high doses. These findings suggest a low toxicity threshold for the studied extract, reinforcing its potential for pharmaceutical applications.

A study conducted by Taofiq et al.evaluated the effects of ethanolic extracts of Ganoderma lucidum and Pleurotus ostreatus on keratinocyte and fibroblast viability at various concentrations. The viability of fibroblasts was maintained at 60% at 100 μg/mL for G. lucidum and at 90% for P. ostreatus. Similar to the trend observed with Ganoderma, the API in the present study resulted in cell viability above 60% at concentrations up to 100 μg/mL, but unlike G. lucidum, it maintained these levels at considerably higher concentrations (up to 1200 μg/mL). Nkadimeng et al.assessed the safety and therapeutic potential of psychedelic mushrooms such as Psilocybe cubensis and Panaeolus cyanescens from the genera Psilocybe and Panaeolus under conditions of pathological hypertrophy. These results indicated that aqueous extracts of P. cyanescens and P. cubensis did not worsen endothelin-1-induced pathological hypertrophy and provided protection against TNF-α-induced injury and cell death at the concentrations tested. With respect to cell viability, the extracts maintained levels above 80% at concentrations of 10 and 25 μg/mL. In the present study, viability was lower at these concentrations but remained close to 80% at concentrations above 50 μg/mL, reaching its peak at 75 μg/mL and slightly decreasing at higher concentrations.

High-performance liquid chromatography (HPLC) was employed to identify and quantify the presence of psilocybin and psilocin in the obtained API. Qualitative identification of the compounds was carried out and confirmed through the injection of analytical standards of psilocybin (PSCB) and psilocin (PSC), along with the API sample, with the absorbance monitored at 266 nm, as shown in Figure. Additionally, UV–Vis spectra were retrieved for the peaks corresponding to PSCB and PSC in the analytical standards, as well as for the peak associated with the compounds detected in the API sample, providing detailed information regarding their UV–Vis absorption profiles, as illustrated in Figure. This approach allowed for comprehensive analysis of the chromatographic behavior of the injected samples, thereby confirming the presence of psilocybin and psilocin in the API.

The retention of the PSCB and PSC standards is consistent with their respective molecular polarity properties, which results in distinct retention times due to their specific interactions with the chromatographic column. Moreover, the chromatogram of the extract shows peaks corresponding to those of the PSCB and PSC standards, indicating the presence of these compounds in the API. The PSCB peaks in the extract appear with moderate intensity, whereas the PSC peak appears as a baseline deviation, suggesting that this compound is present in very low amounts. Additional minor peaks, which do not match those of the standards, may indicate the presence of other compounds in the extract, such as secondary metabolites or polysaccharides. With respect to retention time, the compounds were eluted within the expected range, with only minor variations that do not compromise the reliability of the results. To confirm the presence of these substances in the API, the UV–Vis absorption profiles revealed spectral similarity between the standards and the extract, providing greater confidence in the results presented in Figure.

As shown in Figure, psilocybin exhibited a main absorption band at 223 nm, attributed to the π–π electronic transition in the aromatic systems of its structure, specifically within the indole rings derived from tryptophan. Psilocin also displayed a band at 223 nm, indicating the preservation of the aromatic moiety but with a relatively high absorption intensity. This difference may be explained by the absence of the phosphoryl group in psilocin, which enhances electronic conjugation and increases interaction with UV radiation. Additionally, psilocin exhibited secondary absorption bands between 260 and 300 nm, with peaks at 269, 282, 292, and 297 nm, characteristic of aromatic systems with extended conjugation and the presence of hydroxyl and amine functional groups. On the other hand, the API extract displayed a less intense band at 223 nm, likely due to the low concentrations of psilocybin and psilocin or interference from other matrix constituents, such as proteins, carbohydrates, and secondary metabolites. This interference may obscure the characteristic absorption bands of the target compounds, thereby increasing the complexity of the spectral interpretation of the extract.

HPLC-UV analysis of psilocin and psilocin revealed that the main peaks of these compounds occur between 220–230 nm, whereas secondary bands from 260–300 nm are more prominent in psilocin. However, significant interferences were observed in the crude extracts due to the presence of other matrix components.According to The Merck Index,psilocybin has maximum absorption at 220 nm with secondary absorption at 267 and 290 nm, whereas psilocin has maximum absorption at 222 nm, along with additional peaks at 260, 263, 283, and 293 nm. These values are consistent with the results obtained in the present analysis, in which the standards exhibited an absorption band between 260 and 280 nm. Additionally, a shoulder at 282 nm was observed in the psilocybin standard, a feature not previously reported in The Merck Index,and a band at approximately 292 nm was detected in both standards, corresponding to absorptions at 290 nm for psilocybin and 293 nm for psilocin.

These findings are also supported by the literature, such as the study conducted by Christiansen and Rasmussen, which reported UV–Vis absorption peaks at 219, 266, and 288 nm, as well as a shoulder at 280 nm in the spectra obtained from psilocybin standards. For psilocin standards, the authors identified absorption peaks at 221, 266, 282, and 292 nm. These values corroborate the experimental data obtained in this study, reinforcing the reliability of the results and their consistency with previously published findings. Chromatographic and UV–Vis spectral data (Figures and ) confirmed the presence of psilocybin and psilocin in the API (Active Pharmaceutical Ingredient), enabling the quantification of these compounds in the extract. Quantification of Table was performed via a validated method in accordance with the guidelines established by RDC No. 166/2017, which allows for the determination of compound percentages based on the area under the curve (AUC) of the specific peaks, as described by eqs and .

The API obtained from the extraction of mushrooms belonging to the Psilocybe genus presented an average psilocybin content of approximately 3.26% ± 0.05%, with psilocin levels of approximately 0.34% ± 0.05%. These values correspond to an average of 6.53 mg of psilocybin and 0.69 mg of psilocin per gram of dried mushroom, exceeding the concentrations commonly reported in the literature. According to Stijve and Kuyper, the psilocybin content in Psilocybe mushrooms may range from 0.1 to 1.3% dry weight, whereas psilocin levels are typically lower, varying between 0.01 and 0.35%. Gartz, Allen, and Merlin reported psilocybin concentrations of up to 2% in cultivated species, indicating that cultivation parameters, as well as harvesting and processing conditions, may significantly influence the concentrations of these bioactive alkaloids. Laussmann and Meier-Giebing reported 1.151 ± 0.228 mg of psilocybin and 0.126 ± 0.066 mg of psilocin per 100 mg of dry weight. The elevated psilocybin levels observed in the API may be attributed to multiple factors, including species-specific metabolic profiles, the cultivation environment, the developmental stage at harvest, drying protocols, and extraction methodologies.

Information regarding the solubility of the API in dissolution media within the physiological pH range contributes to the risk assessment associated with biowaiver decisions, using the Biopharmaceutics Classification System (BCS) as a reference criterion. The samples subjected to the solubility assay were quantified via high-performance liquid chromatography (HPLC) through the collection of 1 mL aliquots at time zero (representing the solubility equilibrium condition) and subsequently at 24 and 48 h. Psilocybin and psilocin concentrations were determined in sodium acetate buffer (SAB), sodium phosphate buffer (SPB) and hydrochloric acid (HCl), as shown in Figure.

Chromatographic analysis demonstrated the stability of psilocybin in all three tested media at 0.24 and 48 h intervals. The chromatograms indicated that the concentration of the active compound remained essentially unchanged over time, with only minor fluctuations, thereby indicating its resistance to degradation under the experimental conditions of the solubility assay. Based on eqs and , the concentration of psilocybin in each tested medium (expressed in mg/mL) was used to calculate the dose/solubility ratio (D/S) according to eq. The D/S data enabled the determination of sink conditions, and the results of the solubility analysis are presented in Table.

The results revealed no significant differences in solubility across the tested media for either psilocybin or psilocin. Psilocybin exhibited the highest solubility at pH 4.5 (0.118 mg/mL), with slightly lower values at pH 6.8 (0.115 mg/mL) and pH 1.2 (0.107 mg/mL). Psilocin, which is more apolar than psilocybin, showed lower solubility in all media, with only minor variations among them. Psilocybin demonstrated greater solubility under mildly acidic conditions, suggesting that this pH range may be more suitable for conventional oral formulations, such as capsules and tablets. When compared directly, psilocin is less soluble because of its greater lipophilicity.

The D/S ratios were found to be below the 250 mL threshold, which classifies psilocybin as a high-solubility drug across all tested pH media, according to the Biopharmaceutics Classification System (BCS). Therefore, it can be categorized as a Class I compound with high solubility and high permeability. Additionally, psilocybin recovery data revealed 85.6% in HCl, 94.32% in SAB, and 92.24% in SPB. Although the operational conditions were not optimal, the results suggest that psilocin also has D/S values below 250 mL, indicating that it may likewise be classified as a high-solubility compound.

The sink condition, which assesses whether the volume of solvent available in the body is sufficient to maintain drug dissolution, was recalculated for a 25 mg dose. Typically, dissolution medium volumes of 500, 900, and 1000 mL are used. However, higher volumes may be required for poorly soluble compounds. Considering a volume of 900 mL, the solubility results under sink conditions were determined, as presented in Table.

All sink conditions were greater than 3, indicating sufficient capacity in all cases to ensure complete drug dissolution within typical gastrointestinal fluid volumes. These results demonstrate that, even at its maximum dose (25 mg), psilocybin meets the criteria for high solubility as defined by the Biopharmaceutical Classification System (BCS). However, the variability in sink conditions across different pH values highlights the importance of tailoring formulations to the specific solubility and pH-dependent characteristics of drugs. For the medium containing hydrochloric acid, although the sink condition remained above 3, it represented a moderate sink environmentsufficient to support continuous psilocybin dissolution under acidic conditions, such as those found in the stomach. Nevertheless, the acidic medium had the lowest sink value among the tested media, which suggests that the acidic medium has a relatively limited capacity to maintain excess drug in solution.

The acetate buffer exhibited the highest sink condition among the three media analyzed, with a value of 4.25. This suggests a slightly greater capacity to maintain dissolution equilibrium than the other media do. However, a pH of 4.5 is less representative of physiological conditions within the gastrointestinal tract, as most drug absorption occurs at pH values closer to neutral, such as those found in the small intestine. The phosphate buffer, which simulates the environment of the small intestine, presented a sink condition of 4.14compared with that of the acetate buffer. This result is particularly relevant, as the small intestine is the primary site of psilocybin absorption and its conversion to psilocin, the active metabolite. The moderately high solubility observed at this pH suggests that phosphate buffer would be an ideal medium for dissolution testing, as it more accurately reflects physiological conditions.